Research Article iMedPub Journals Journal of Rare Disorders: Diagnosis & Therapy 2016 http://www.imedpub.com/ ISSN 2380-7245 Vol. 2 No. 5: 20 DOI: 10.21767/2380-7245.100049 Biochemical Characterisation and Whole Zandberg L1, Erasmus E1, Suormala T2,3, Mienie LJ1, Genome Expression Profiling of Cultured Skin Mels CMC4 and Van Dijk AA1 Fibroblasts from Two South African Adults 1 Biochemistry, Focus Area Human with Urinary 3-Hydroxyisovaleric Acid and Metabolomics, North-West University, 3-Methylcrotonylglycine Potchefstroom, South Africa 2 Division of Metabolism, University Children's Hospital, Zurich, Switzerland 3 Metabolic Unit, University Children's Abstract Hospital, Basel, Switzerland 4 Hypertension in Africa Research Team, We report on the first case of marginal 3-methylcrotonyl-CoA carboxylase North-West University, Potchefstroom, (MCC) deficiency in South Africa. Urinary 3-hydroxyisovaleric acid and South Africa 3-methylcrotonylglycine were detected in four males of a non-consanguineous family. Only the index patient (NWU001) had non-specific symptoms, the others were asymptomatic. The inherited metabolite profile and partially reduced MCC Corresponding author: activity were indicative of marginal MCC deficiency. In vivo L-leucine loading Van Dijk AA confirmed a reduced flux through the leucine degradation pathway. No known deleterious mutations were detected in the open reading frames of MCCC1 and [email protected] MCCC2. NWU001 was heterozygous for a SNP in MCCC1 (rs2270968; c.1391A>C, p.H464P). NWU002 was heterozygous for a MCCC2 splice variant which skips exon Biochemistry Division, North-West 7 and causes an in frame deletion of 38 amino acids that is identical to a predicted University, Private Bag X6001 Noordbrug, shorter MCCC2 isoform-2 (Q9HCC0-2). Whole genome expression profiles from 2520 Potchefstroom, South Africa. cultured skin fibroblasts of NWU001 and NWU002 and two healthy adults using Affymetrix® HuExST1.0 arrays detected 14237 significantly differentially expressed Tel: +27 18 299 2317 transcript IDs of which only 1277 have known annotation and gene association. Fax: +27 18 299 2316 The underlying molecular interactions, secondary signalling responses and functional relationships of these 1277 transcripts were inspected following a knowledge-based functional analyses approach using Ingenuity Pathway Analysis software. The transcriptome had a footprint of oxidative stress, disruption of Citation: Zandberg L, Erasmus E, Suormala energy homeostasis, inflammation, impaired cellular maintenance and repair T, et al. Biochemical Characterisation mechanisms. Of note was the significant up regulation of the fatty acid amide and Whole Genome Expression hydrolase variant 2 (FAAH2) HuChrX transcript in the anandamide degradation Profiling of Cultured Skin Fibroblasts from Two South African Adults with canonical pathway. The observations that the two MCC transcripts were not Urinary 3-Hydroxyisovaleric Acid and significantly differentially expressed and that more than 90% of the significantly 3-Methylcrotonylglycine. J Rare Disord differently expressed transcripts are still poorly annotated further support Diagn Ther. 2016, 2:5. the notion that secondary factors other than the MCC loci impact on the MCC deficiency phenome. Keywords: 3-methylcrotonyl-CoA carboxylase deficiency; Oxidative stress; Affymetrix® HuExST1.0 array; 3-Hydroxyisovaleric acid; 3-Methylcrotonylglycine; Leucine catabolism Received: April 06, 2016; Accepted: September 18, 2016; Published: September 21, 2016 Introduction diseases [1-5]. This is evident from the often broad spectrum of clinical symptoms and highly variable time of onset for the same There is an increasing awareness that although inborn errors of disease [4,6-10]. One such disease is isolated 3-methylcrotonyl- metabolism are considered simple monogenic disorders caused by a deficiency of a single enzyme, they often act as complex CoA carboxylase (MCC) deficiency (MIM #210200 and MIM © Under License of Creative Commons Attribution 3.0 License | This article is available from: //www.raredisorders.imedpub.com/ 1 Journal of Rare Disorders: Diagnosis & Therapy 2016 ISSN 2380-7245 Vol. 2 No. 5: 20 #210210) [11-14]. MCC deficiency is an autosomal recessive enzymatic studies andin vivo L-leucine loading suggest that these disorder of the leucine catabolism which usually presents with individuals have marginal MCC deficiency. Intriguingly, the whole elevated levels of urinary 3-hydroxyisovaleric acid (HIVA) and genome expression profile of cultured skin fibroblasts indicated 3-methylcrotonylglycine (MCG) [15-16]. Newborn screening that both MCCC1 and MCCC2 were not significantly differentially (NBS) revealed that MCC deficiency is the most frequently expressed in cells of the affected individuals compared to the detected organic aciduria [13,17,18]. MCC deficiency is regarded controls. The gene expression profiling and inheritance pattern as a disease with a low penetrance [19] since less than 10% in this family seem to suggest that the chromosome-X (HuChrX) of individuals with a metabolite profile indicative of MCC might impact on the pathomechanism of this disorder. deficiency develops [14]. The clinical, biochemical, and enzymatic characterization of MCC deficiency have been well documented, Clinical Presentation but the pathomechanism remains largely unknown [9,15,20-26]. The index patient (NWU001), a Caucasian male from a non- MCC is one of four biotin-dependent carboxylases and is consanguineous South African family, was 48 years of age when comprised of two non-identical subunits, alpha (MCCA) and beta he presented with mild hepatosplenomegaly. "The metabolic screen revealed elevated levels of urinary 3-hydroxyisovaleric (MCCB), which forms a α6β6 hetero-dodecamer [27-29]. The two subunits are encoded by nuclear genes and have signal peptides acid and 3-methylcrotonylglycine which is usually indicative that target them to the mitochondrion [30]. The alpha subunit of MCC deficiency." He was advised to report for metabolic is encoded by the MCCC1 (formerly called MCCA) gene located screening. The patient mentioned a 20 year history of frequently on chromosome 3q25-q27 and has 19 coding exons. The beta occurring symptoms often in association with episodes of subunit is encoded by the MCCC2 (formerly called MCCB) gene chronic fatigue. He reported reduced tolerance to exercise and frequently experienced acute muscle cramps which subsided located on chromosome 5q12-q13 and has 17 coding exons [31- with rest. He complained of general weakness, poor tolerance 33]. About 132 disease-causing mutations have been reported to environmental factors and being prone to hay fever, eczema, for the MCCC1 and MCCC2 genes [19] but no prevalent mutation mouth ulcers and sinusitis. Although the patient had a body has been identified and there is no clear genotype-phenotype mass index of 27.9 (Obese reference range: >26.9), he appeared correlation [17,21,31]. The MCC activity of patients with healthy and not overweight. His blood chemistry was mostly deleterious homozygous or compound heterozygous mutations within normal ranges, with the exception of a slightly elevated is usually reduced to <5% of the mean activity of the controls alanine aminotransferase activity of 41 U/L (Male age 12 [15,21,22,31], but a few cases have been reported with residual months-60 years reference range: 10-40 U/L) and elevated levels MCC activity, up to 34% [19,22,26] and some individuals with of 784 pmol/L VitB12 (Normal reference range: 148 - 616 pmol/L). urinary metabolites indicative of MCC deficiency who only have His total cholesterol was borderline high, 5.5 mmol/L (Borderline a single heterozygous MCC mutation have almost normal MCC high reference range: 5.18-6.19 mmol/L) and he had low HDL, activity [17]. There is an increasing awareness of the difficulty to 0.4 mmol/L (Normal reference range: <1.04 mmol/L), normal diagnose MCC deficiency because some patients present with LDL, 2.67 mmol/L (Normal reference range: <2.85 mmol/L) but an atypical metabolic profile with only trace amounts or even very high levels of triglycerides, 6.73 mmol/L (normal reference no detectable urinary MCG [22], the variability and non-specific range: <5.65 mmol/L) [35]. He presented with normal blood nature of its symptoms and the growing number of individuals pressure, fasting glucose and haematological parameters. presenting with MCC deficiency associated metabolites but with The second family member, NWU002 (male, age 52), sibling of only a single mutation inMCCC1 or MCCC2 [17]. Recently, analyses the index patient NWU001 had no clinical symptoms related to of the first exome sequence data from MCC deficiency cases [34] that of his brother. However, he also presented with a similar revealed that individuals who have the same mutation most likely profile of abnormal urinary organic acids and acylcarnitine share a common ancestor, that rare mutations outside of MCC urinary metabolites as NWU001. At the time of investigation genes can cause non-specific phenotypes hitherto attributed NWU002 already received medication to control persistent high to MCC deficiency in genetically closely related patients and levels of total cholesterol and his total cholesterol level was 4.9 that the clinical consequences of MCC deficiency could be less mmol/L (Reference range: 2.8-5.2 mmol/L). pervasive than generally accepted. The authors also stated that further studies should be done to assess the value of treating In addition to the index patient